Mitochondrial diseases are devastating disorders for which there is no cure and no proven treatment. About 1 in 2000 individuals are at risk of developing a mitochondrial disease sometime in their lifetime. Half of those affected are children who show symptoms before age 5 and approximately 80% of these will die before age 20. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much work is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genome, as point mutations, deletions, rearrangements, or depletion of the mitochondrial DNA (mtDNA). The mutation rate of the mitochondrial genome is 10-20 times greater than of nuclear DNA, and mtDNA is more prone to oxidative damage than is nuclear DNA. Mutations in human mtDNA cause premature aging, severe neuromuscular pathologies and maternally inherited metabolic diseases, and influence apoptosis. The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3'-5' exonuclease proofreading activity, and 5'dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, pol gamma participates in DNA replication and DNA repair. The 140 kDa catalytic subunit for pol gamma is encoded by the nuclear POLG gene. To date nearly 250 pathogenic mutations in POLG that cause a wide spectrum of disease including Progressive external ophthalmoplegia (PEO), parkinsonism, premature menopause, Alpers syndrome, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO). We previously reported on a patient, a infant-old boy who presented with hepatic failure, and was found to have severe mtDNA depletion in liver and muscle. Whole-exome sequencing identified a homozygous missense variant (c.544C > T, p.R182W) in the accessory subunit of mitochondrial DNA polymerase gamma (POLG2), which is required for mitochondrial DNA replication. This variant is predicted to disrupt a critical region needed for homodimerization of the POLG2 protein and cause loss of processive DNA synthesis. Both parents were phenotypically normal and heterozygous for this variant. Heterozygous mutations in POLG2 were previously associated with progressive external ophthalmoplegia and mtDNA deletions. This is the first report of a patient with a homozygous mutation in POLG2 and with a clinicalpresentation of severe hepatic failure and mitochondrial depletion. In a new publication, we characterized this homozygous R182W p55 mutation using in vivo cultured cell models and in vitro biochemical assessments. Compared to control fibroblasts, homozygous R182W p55 primary dermal fibroblasts exhibit a two-fold slower doubling time, reduced mtDNA copy number and reduced levels of POLG and POLG2 transcripts correlating with the reported disease state. Expression of R182W p55 in HEK293 cells impairs oxidative-phosphorylation. Biochemically, R182W p55 displays DNA binding and association with p140 similar to WT p55. R182W p55 mimics the ability of WT p55 to stimulate primer extension, support steady-state nucleotide incorporation, and suppress the exonuclease function of Pol in vitro. However, R182W p55 has severe defects in protein stability as determined by differential scanning fluorimetry and in stimulating function as determined by thermal inactivation. These data demonstrate that the Chr17: 62492543G>A mutation in POLG2, R182W p55, severely impairs stability of the accessory subunit and is the likely cause of the disease phenotype. Mitochondrial single-stranded DNA binding protein (mtSSB) is an essential component of the human mtDNA replication machinery. We utilized single molecule methods to examine the mode by which human mtSSB binds DNA to define how mtSSB may interact with the mtDNA replication fork and influence the activities of other mtDNA metabolizing enzymes. Direct visualization of individual mtSSB molecules and estimation of volume by atomic force microscopy confirmed the tetrameric conformation of human mtSSB. The equilibrium binding affinity and specificity of mtSSB for single-stranded DNA were determined by fluorescence methods. AFM imaging revealed a random distribution of mtSSB tetramers bound to extended regions of single-stranded DNA, strongly suggesting non-cooperative binding by mtSSB. Selective binding of mtSSB to single-stranded DNA was confirmed by AFM imaging of individual mtSSB tetramers bound to gapped plasmid DNA substrates bearing defined single-stranded regions. Shortening of the contour length of gapped DNA upon binding mtSSB was attributed to DNA wrapping around mtSSB. Tracing the DNA path in mtSSB-ssDNA complexes with Dual Resonance frequency Enhanced Electrostatic force Microscopy (DREEM) established a single binding mode in which one DNA strand winds only once around each mtSSB tetramer. These results suggest mtSSB does not saturate or fully protect single-stranded replication intermediates during mtDNA synthesis, leaving the mitochondrial genome vulnerable to chemical mutagenesis, deletions driven by primer relocation, or other actions consistent with clinically observed deletion biases. The base excision repair (BER) pathway is the primary pathway involved in maintaining the integrity of mtDNA. Several enzymes that participate in BER within the nucleus have also been identified in the mitochondria. The nei-like (NEIL) DNA glycosylases initiate BER by removing oxidized pyrimidine bases and others such as the ring-opened formamidopyrimidine lesions. During BER the NEIL enzymes interact with several proteins that are required for DNA replication and transcription. In collaboration with Aishwarya Prakash (U South Alabama) we detected NEIL1 in purified mitochondrial extracts from human cells and utilized protein painting techniques, far-western analysis, and gel-filtration chromatography to establish that NEIL1 interacts with the human mitochondrial single-stranded DNA binding protein (mtSSB) via its C-terminal tail. Work is ongoing to scrutinize the NEIL1-mtSSB interaction in the presence of a partial-duplex DNA substrate and generate low-resolution molecular structures using small-angle X-ray scattering (SAXS).